Figure 3
Interaction between TIF1β and HP1β is compromised by phosphorylation of TIF1β/Ser473. (A) Schematic representation of domain organization of TIF1β protein. The position of Ser473 relative to the PXVXL motif is shown. (B) Western blot results of co-immunoprecipitation of over-expressed FLAG-TIF1β and HA-HP1β. FLAG-TIF1β/S473A, FLAG-TIF1β/S473E and wild-type FLAG-TIF1β were co-expressed with HA-HP1β into 293T cells. The level of ectopically expressed FLAG-TIF1βs (Input, M2) or M2-beads immunoprecipitated FLAG-TIF1βs (M2-IP, M2) was probed with M2 antibodies (TIF1β, M2). The level of ectopically expressed HA-HP1β (Input, HA) or M2-beads co-immunoprecipitated HA-HP1βs (M2-IP, HA) was probed with monoclonal anti-HA antibodies (HP1β, HA). (C) In vitro GST pull-down assay of TIF1βs by recombinant GST-HP1β. Wild-type and mutant FLAG-TIF1βs were expressed in 293T cells. These FLAG-TIF1βs were immunoprecipitated by M2 beads and eluted with M2 peptide. The levels of FLAG-TIF1βs from 1/10 input for each pull down assay were shown (lanes 8–10). Eluted FLAG-TIF1βs were incubated with GST-beads alone (lanes 1–3) or incubated with recombinant GST-HP1β bound glutathione-beads (lanes 4–6). The levels of GST-HP1β and FLAG-TIF1βs in each pull down assay were shown (lanes 1–6). Quantitation of the Western blots (by ImageGauge software) from each pull down sample (lanes 4–6) showing the relative level of FLAG-TIF1βs (A, TIF1β/S473A; E, TIF1β/S473E; W, wild-type TIF1β) pulled down by GST-HP1β, after normalization to individual GST-HP1β levels (right panel). (D) Interaction between in vitro translated FLAG-TIF1βs and HA-HP1β. Upper panel, In vitro translated [35S] FLAG-TIF1βs and [35S]-HA-HP1β were incubated for 30 min before being immunoprecipitated by M2 beads in the presence of 0.5 M NaCl. The immunoprecipitates were resolved by SDS-PAGE and followed by autoradiography to visualize HA-HP1β (upper right panel, M2-pull down). Lower panel, In vitro translated [35S] FLAG-TIF1βs and [35S] HA-HP1β were pulled down by protein-G coupled anti-HA antibody. The immunoprecipitated FLAG-TIF1βs are shown in the lower right panel (TIF1βs, IP). Supernatants after immunoprecipitation are also shown, as indicated (S).